The long term goal of this project is to determine the molecular basis for cell-specific regulation ofpre-mRNA alternative splicing. Alternative splicing plays a prominent role in generating the diversity of the human proteome. A large fraction of alternatively spliced pre-mRNAs undergo cell-specific regulation in which splicing patterns are modulated according to cell type, developmental stage, or in response to external stimuli. Regulation of alternative splicing directs determinative long term and short term cellular events. Human disease can result from altered splicing regulation and mis-expression of splicing patterns that are inappropriate for adult tissues. Despite the importance of splicing regulation to normal and abnormal cellular function, little is known about the mechanisms that determine cell-specific splicing patterns. We will continue investigating regulation of alter native splicing during skeletal and cardiac muscle development using the cardiac troponin T gene. Three families of splicing regulators have been shown to bind to previously defined cis-acting elements and effect positive or negative modulation: CUG-BP and ETR-3 like factors (CELF), muscle blind (MBNL), and polypyrimidine tract binding protein (PTB). In this proposal, we will define the CELF and MBNL protein domains required for splicing regulation. We will use an existing cell-free splicing assay to identify the molecular interactions that ultimately link intronic cell-specific regulatory elements to the basal splicing machinery to activate splicing. Additional pre-mRNA targets for these regulators have been identified and will be utilized for comparative analysis of activation pathways. We will also investigate the contributions of endogenous regulators to the modulation and coordination of multiple splicing transitions that occur during C2C12 differentiation. This investigation will enhance the understanding of this critical point of gene regulation and establish a foundation to develop approaches to reverse or circumvent pathological processes. [unreadable] [unreadable]